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Endocrinology Vol. 144, No. 6 2304-2310
Copyright © 2003 by The Endocrine Society

Activation of Cell Adhesion Kinase ß by Mechanical Stretch in Vascular Smooth Muscle Cells

Hiroaki Iwasaki, Takanobu Yoshimoto, Toru Sugiyama and Yukio Hirata

Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Tokyo 113-8519, Japan

Address all correspondence and requests for reprints to: Yukio Hirata, M.D., Ph.D., Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail: yhirata.cme{at}tmd.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have studied whether activation of cell adhesion kinase ß (CAKß) is involved in stretch-induced signaling pathway in cultured rat vascular smooth muscle cells. Cyclic stretch (1 Hz) induced a rapid (within 1 min) phosphorylation of CAKß, whose effect was time and strength dependent. Both Ca2+ and Na+ ionophores (A23187 and monensin) stimulated phosphorylation of CAKß in a similar fashion to mechanical stretch. The stretch-induced phosphorylation of CAKß was inhibited completely by an intracellular Ca2+ chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)] and largely by gadolinium, but only partially by an extracellular Ca2+ chelator (EGTA). An angiotensin type 1 receptor antagonist (CV11974) abolished the phosphorylation of CAKß stimulated by angiotensin II, but not by mechanical stretch. Mechanical stretch rapidly (within 1 min) increased the association of CAKß with c-Src, but not pp125focal adhesion kinase. Stretch-induced phosphorylation of ERK1/2 was inhibited by EGTA and an inhibitor of the Src kinase family [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], but not by cytochalasin D, to disrupt actin polymerization. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or cytochalasin D did not affect stretch-induced phosphorylation of CAKß. These data suggest that mechanical stretch stimulates activation of CAKß, followed by its association with c-Src, which requires ion influx mainly via stretch-activated nonselective ion channels, thereby leading to activation of the p21Ras/ERK1/2 cascade in vascular smooth muscle cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYPERTENSION increases hemodynamic force in vascular wall, which is a well recognized risk factor for stroke, ischemic heart disease, peripheral vascular disease, and progressive renal disease (1, 2, 3). Mechanical stretch and circumferential tension experienced in vascular smooth muscle cells (VSMC) are directly associated with intimal-media thickness of common carotid artery in vivo (4, 5), suggesting that mechanical stretch by pressure overload also contributes to the pathogenesis of atherosclerosis. Several in vitro studies using cyclic stretch have revealed that excessive stretching of VSMC can provoke various intracellular signal events, including an increase in the intracellular Ca2+ concentration ([Ca2+]i) to stimulate a number of downstream signaling pathways, such as p21Ras and MAPK (6).

There are two mechanisms for intracellular Ca2+ mobilization; one is Ca2+ influx through Ca2+-permeable channels, and another is Ca2+ release from intracellular Ca2+ stores. It has been shown that gadolinium (Gd), a cation-selective, stretch-activated (SA) channel blocker, can block the stretch-induced Ca2+ response (7), suggesting that the increase in [Ca2+]i in response to stretching is via the SA channel. SA channels have been demonstrated in a variety of cells by the single-channel, patch-clamp technique; however, their physiological significance is as yet unclear. Recently, we demonstrated that stretch-induced Ca2+ influx via Gd-sensitive SA channels contributed to the early signaling events to stimulate ERK1/2, thereby leading to VSMC growth (8).

It has been proposed that signaling pathways converging at receptor and/or nonreceptor tyrosine kinases play central roles in mitogenesis, differentiation, and migration. Cell adhesion kinase-ß (CAKß), also known as proline-rich tyrosine kinase 2, related adhesion focal tyrosine kinase, or Ca2+-dependent tyrosine kinase, is a member of cytoplasmic tyrosine kinase family and shows a high sequence homology to the pp125focal adhesion kinase (pp125FAK) (9). Despite the partial difference in amino acid residues in the C-terminal regions, the differential subcellular localization and the association with the cytoskeleton proteins shared by CAKß and FAK are consistent with their different regulations (10). In fact, activation of CAKß is more rapidly influenced by humoral factors such as G protein-coupled receptor agonists, protein kinase C activation, and increase in [Ca2+]i (9), whereas FAK is slowly activated and primarily controlled by mechanical factors, such as cell adhesion, shear stress, and stretching (10, 11). However, as CAKß expression was increased in FAK-null cells as partial compensation for FAK function (12), CAKß may be a candidate for the key signaling molecule in the mechanism of mechanotransduction in VSMC.

Here we show that mechanical stretch stimulates activation of CAKß and its association with c-Src, which requires ion influx mainly through SA channels in rat VSMC.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Monensin, A23187, lysophosphatidic acid (LPA), phorbol 12-myristate 13-acetate (PMA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM), EGTA, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), and cytochalasin D were purchased from Calbiochem-Novabiochem (La Jolla, CA). Angiotensin II (AngII) was obtained from Peptide Institute (Osaka, Japan). Polyclonal antiphospho-CAKß(pY402), and monoclonal anti-FAK antibodies were purchased from BioSource International, Inc. (Camarillo, CA). Polyclonal anti-CAKß and polyclonal anti-ERK2 antibodies and protein A/G-agarose were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Monoclonal antiphosphotyrosine (pTyr) (PY20) and monoclonal anti-CAKß antibodies were obtained from Transduction Laboratories, Inc. (Lexington, KY). Monoclonal and polyclonal anti-CAKß, monoclonal anti-Src (GD11), and monoclonal anti-FAK antibodies were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antiphospho ERK1/2 antibody was purchased from New England Biolabs, Inc. (Beverly, MA). Antimouse and antirabbit immunoglobulin G horseradish peroxidase-conjugated second antibodies were obtained from Amersham International (Little Chalfont, UK), and Gd chloride was purchased from Wako Chemicals (Osaka, Japan). The angiotensin type 1 (AT1) receptor antagonist (CV11974) was a gift from Takeda Pharmaceutical Co. (Tokyo, Japan).

Cell culture
VSMCs were prepared from the thoracic aorta of 12-wk-old male Sprague Dawley rats by the explant method and cultured in DMEM containing 10% fetal calf serum at 37 C in a humidified atmosphere of 95% air-5% CO2 as previously described (8). Quiescent VSMC (5–15th passages) after 48–72 h of serum starvation were used in the following experiments.

Cyclic strain
Cells were grown to confluence (1 x 105 cells/well) in six-well silicone Elaster-bottom culture plates with a hydrophilic surface (FLX-P1001C; Flexcell Corp., McKeesport, PA). Cells were subjected to mechanical deformation with the Flexcell strain unit (model FX-2000) (13, 14). Negative pressure by vacuum was repetitively applied (1 Hz, 0.5 sec on-time) to the rubber-bottomed dishes via the baseplate, and they were placed in a humidified incubator with 5% CO2 at 37 C as previously reported (8). This system produces a gradient of deformation across the membrane at the bottom of the plates, with the highest strain at the periphery and the lowest strain at the center of the dishes. The maximal deformations were 6.25%, 12.5%, and 25% at -5, -9, and -21 kPa, respectively. Cells on the entire rubber-bottomed surface were harvested in each experiment,

Immunoblotting and immunoprecipitation
Immunoblotting was performed as previously described (8). After cells were subjected to mechanical stretch for the indicated times, medium was replaced with 100 µl SDS-PAGE buffer (62.5 mM Tris-HCl, 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue), pH 6.8. After brief sonication, samples were boiled for 5 min at 95 C and centrifuged, and aliquots of the supernatant were subjected to 10% SDS-PAGE. Proteins in the gel were transferred to a nitrocellulose membrane by electroblotting. The membranes were treated with anti-pTyr (1:2000), anti-CAKß (1:1000), anti-phospho CAKß(pY402) (1:2000), antiphospho-ERK1/2 (1:1000), anti-ERK2 (1:5000), and anti-c-Src (1:3000) antibodies, respectively, followed by second antibodies conjugated with horseradish peroxidase (1:2000); immunoreactive proteins were detected by an enhanced chemiluminescence system (Amersham International).

Immunoprecipitation of CAKß and FAK was performed as previously described (15). In brief, cells after mechanical stretch were lysed in 0.8 ml lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 25 mM EDTA, 1.0% Triton-X, 0.1% sodium dodecyl sulfate, 10% glycerol, 100 mM NaF, 100 mM Na3P2O7, 1.0% deoxycholic acid, 1 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, and 10 µg/ml aprotinin), pH 7.4. After the lysates were sonicated and centrifuged, the supernatants were rocked with either polyclonal anti-CAKß or anti-FAK antibody (2 µg/ml) with protein A/G agarose for 16 h at 4 C. The beads were washed three times with lysis buffer, solubilized in Laemmli sample buffer, and subjected to the immunoblotting. A representative of two or three experiments is presented in each figure.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cyclic stretch time- and strength-dependently activates CAKß
To determine whether cyclic stretch of VSMC induces activation of CAKß in a time- and strength-dependent manner, CAKß activation was assessed by immunoprecipitation with anti-pTyr antibody, followed by immunoblotting with anti-CAKß antibody and immunoblotting with the polyclonal antibody that selectively recognizes phosphorylated tyrosine 402 residue of CAKß (pY402). Mechanical stretch (-21 kPa) rapidly and transiently caused phosphorylation of CAKß, which peaked at 1 min and then decreased by 5 min (Fig. 1Go, A and B). The phosphorylation of CAKß by stretching (1 min) was strength dependent (-5, -9, and -21 kPa); a minimum response was induced by as little as -5 kPa and a maximal response by -21 kPa of cyclic stretch (Fig. 1Go, C and D). Therefore, subsequent experiments were performed by cyclic stretch of -21 kPa for 1 min unless otherwise stated.



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  		Figure 1. Time- and strength-dependent activation of CAKß by mechanical stretch in rat VSMC. Cells were stimulated with cyclic stretch (-21 kPa) for the indicated times (A and B) or with the indicated strength for 1 min (C and D). Cell lysates were immunoprecipitated with anti-pTyr antibody and then immunoblotted with anti-CAKß antibody (A and C) or immunoblotted with anti-phospho CAKß(pY402) antibody (upper panel) and anti-CAKß antibody (lower panel), respectively (B and D). An arrowhead indicates tyrosine-phosphorylated CAKß.

 
Ca2+ and Na+ ionophores activate CAKß
Previous studies revealed that mechanical stretch caused an increase in [Ca2+]i via Na+-permeable SA channels (16). To determine whether accumulation of either intracellular Ca2+ or Na+ contributes to activation of CAKß, we examined the phosphorylation of CAKß and ERK1/2 after treatment with Ca2+ and Na+ ionophores. Both a Ca2+ ionophore (A23187, 10 µM) and a Na+ ionophore (monensin, 10 µM) caused greater phosphorylation of CAKß (Fig. 2AGo) and ERK1/2 (Fig. 2BGo) than did mechanical stretch. These data suggest that accumulation of either intracellular Ca2+ or Na+ is sufficient for CAKß and ERK1/2 activation, and mechanical stretch-stimulated CAKß and ERK1/2 may be due to increases in [Ca2+]i or the intracellular Na+ concentration ([Na+]i) via SA channels.



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  		Figure 2. Effects of mechanical stretch and ion ionophores on phosphorylation of CAKß and ERK1/2. Cells were stimulated by stretch (-21 kPa), A23187 (10 µM), or monensin (10 µM) for 1 min (A) or 5 min (B). A, Cell lysates were immunoprecipitated with anti-CAKß antibody and then immunoblotted with anti-pTyr antibody (upper panel) and anti-CAKß antibody (lower panel), respectively. An arrowhead indicates tyrosine-phosphorylated CAKß. B, Cell lysates were subjected to immunoblotting with antiphospho ERK1/2 antibody (upper panel) and anti-ERK2 antibody (lower panel), respectively. Arrowheads indicate phosphorylated ERK1/2.

 
An increase in [Ca2+]i is required for activation of CAKß
A previous study revealed that an increase in [Ca2+]i was a pivotal signal for activation of CAKß by AngII in VSMC (17). The initial rapid and transient rise of [Ca2+]i in response to stretching is attenuated by either EGTA or Gd (18). To confirm that an increase in [Ca2+]i by ion influx is responsible for CAKß activation by mechanical stretch, we examined the effects of cell-permeable (BAPTA-AM) and noncell-permeable (EGTA) calcium chelators on the stretch- and LPA-induced phosphorylation of CAKß. Pretreatment with BAPTA-AM (50 µM) completely blocked the phosphorylation of CAKß by mechanical stretch, but not by LPA (100 nM; Fig. 3Go, A, C, and D), whereas pretreatment with EGTA (5 mM) only partially inhibited the phosphorylation of CAKß after stretching, but not after treatment with LPA (Fig. 3Go, B–D); neither BAPTA-AM nor EGTA alone affected the basal CAKß level. We have previously shown that the stretch-induced phosphorylation of ERK1/2 was abolished by an intracellular Ca2+ antagonist (TMB-8) (8) and decreased by EGTA (5 mM; data not shown). Taken together, our data suggest that accumulation of intracellular Ca2+ partly mediated by Ca2+ influx from the extracellular sources is involved in CAKß activation.



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  		Figure 3. Effects of Ca2+ chelators on stretch- and LPA-induced phosphorylation of CAKß. Cells pretreated with or without BAPTA (A; 50 µM) for 30 min or EGTA (B; 5 mM) for 2 min were stimulated with mechanical stretch (-21 kPa) or LPA (100 nM) for 1 min. Cell lysates were immunoprecipitated with anti-CAKß antibody and then immunoblotted with anti-pTyr antibody (upper panel) and anti-CAKß antibody (lower panel), respectively. An arrowhead indicates tyrosine-phosphorylated CAKß. C and D, The effects of BAPTA and EGTA on stretch (C)- or LPA (D)-induced CAKß phosphorylation as evaluated by densitometric analysis are shown. Each column is corrected as the percentage of those calculated from mean values of two different experiments without Ca2+ chelators.

 
Stretch-induced activation of CAKß is mediated via Gd-sensitive SA ion channel, but not via AngII receptors
To determine the exact route for accumulation of [Ca2+]i by stretching, we examined the effect of Gd, an inhibitor of SA ion channels, on the stretch-induced phosphorylation of CAKß. Pretreatment with Gd inhibited the stretch-induced phosphorylation of CAKß in a dose-dependent manner (10–50 µM); Gd alone had no effect on basal CAKß level (Fig. 4AGo and data not shown). We have previously reported that Gd completely blocked the stretch-induced phosphorylation of ERK1/2 (8). Taken together, our present data suggest that Gd-sensitive SA ion channels are largely responsible for CAKß activation and subsequent ERK1/2 activation. It should be also mentioned that pretreatment with a high dose of Gd did not completely inhibit CAKß, suggesting that there may be an alternative CAKß activation pathway that does not depend on SA channel activation.



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  		Figure 4. Effects of Gd and AT1 receptor antagonist on stretch-induced phosphorylation of CAKß. Cells pretreated with or without Gd (10–50 µM) for 60 min (A) or CV11974 (1 µM) for 30 min (B) were stimulated with mechanical stretch (-21 kPa) or AngII (100 nM) for 1 min. Cell lysates were immunoprecipitated with anti-CAKß antibody and then immunoblotted with anti-pTyr antibody. An arrowhead indicates tyrosine-phosphorylated CAKß.

 
Mechanical stretch has shown to release AngII and activate ERK1/2 in an autocrine/paracrine manner in cardiomyocytes (19). Recently, we demonstrated that AngII stimulated phosphorylation of CAKß via an increase in [Ca2+]i in rat VSMC (17). To exclude the involvement of autocrine/paracrine-secreted AngII in stretch-activated pathway, we examined the effect of an AT1 receptor antagonist (CV11974) on the stretch-induced phosphorylation of CAKß. Pretreatment with CV11974 (1 µM), which markedly blocked phosphorylation of CAKß stimulated by AngII (100 nM), failed to do so after mechanical stretch (Fig. 4BGo). Therefore, it is unlikely that autocrine/paracrine secretion of AngII after stretching is involved in the stretch-induced activation of CAKß in VSMC.

Mechanical stretch stimulates association of CAKß with c-Src
A major autophosphorylation site (Tyr402) of CAKß is recognized by tyrosine kinases containing the SH2 domains, such as c-Src and Fyn. Binding of c-Src to this residue is necessary for phosphorylation of Shc and its association with Grb2, thereby linking to the subsequent activation of ERK1/2 cascades (20, 21). To determine whether mechanical stretch enhances the formation of a signaling complex between CAKß and c-Src, we performed immunoprecipitation with anti-CAKß antibody followed by immunoblotting with either anti-CAKß or anti-c-Src antibody. Mechanical stretch rapidly (within 1 min) stimulated the association of CAKß with c-Src (Fig. 5AGo) in a time course similar to that of tyrosine phosphorylation of CAKß (Fig. 1AGo). Furthermore, pretreatment with PP2 (10 µM), a selective inhibitor of Src kinase family (22), inhibited the stretch-induced phosphorylation of ERK1/2, whereas PP2 had no effect on the PMA (1 µM)-induced phosphorylation of ERK1/2 (Fig. 5BGo). In addition, PP2 did not affect stretch-induced CAKß tyrosine phosphorylation (Fig. 5CGo). These data suggest that mechanical stretch is responsible for the formation of signaling complex between CAKß and c-Src, thereby possibly recruiting adaptor molecules, such as Shc and Grb2.



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  		Figure 5. Association of c-Src with CAKß by mechanical stretch. A, After stimulation with mechanical stretch (-21 kPa) for the indicated times, cell lysates were immunoprecipitated with anti-CAKß antibody and then immunoblotted with anti-c-Src antibody (upper panel) and anti-CAKß antibody (lower panel), respectively. An arrowhead denotes c-Src (upper panel) and CAKß (lower panel), respectively. B, Cells pretreated with or without PP-2 (10 µM) for 15 min were stimulated with mechanical stretch or PMA (1 µM) for 5 min and then subjected to immunoblotting with antiphospho ERK1/2 antibody (upper panel) and anti-ERK2 antibody (lower panel), respectively. Arrowheads indicate phosphorylated ERK1/2. C, The effect of PP-2 on stretch-induced phosphorylation of CAKß. Cells pretreated with or without PP-2 (10 µM) for 15 min were stimulated with mechanical stretch (-21 kPa) for 1 min. Cell lysates were immunoprecipitated with anti-CAKß antibody and then immunoblotted with anti-pTyr antibody (upper panel) and anti-CAKßantibody (lower panel), respectively. An arrowhead indicates tyrosine-phosphorylated CAKß.

 
Stretch-induced activation of CAKß and ERK1/2 are independent of actin cytoskeleton and FAK
Recently, it was reported that histamine, a G protein-coupled receptor agonist, induced the translocation of CAKß to focal adhesions and enhanced the phosphorylation of CAKß and ERK1/2 in HeLa cells, whose effects were abolished by cytoskeletal disassembly with cytochalasin D (23). To determine whether mechanical stretch contributes to focal adhesion-dependent ERK1/2 activation, we examined the effect of cytochalasin D, an actin polymerization inhibitor, on stretch-induced activation of ERK1/2. Cytochalasin D (5 µM) had no effect on stretch-induced phosphorylation of ERK1/2 (Fig. 6AGo), suggesting that stretch-induced CAKß phosphorylation and ERK1/2 activation are independent from the integrity of the actin cytoskeleton. In addition, cytochalasin D had no effect on stretch-induced phosphorylation of CAKß (Fig. 6CGo).



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  		Figure 6. Effects of mechanical stretch on involvement of actin cytoskeleton and association of FAK with CAKß. A, Cells pretreated with or without cytochalasin D (5 µM) for 30 min were stimulated with mechanical stretch (-21 kPa) for 5 min and then subjected to immunoblotting with antiphospho-ERK1/2 antibody (upper panel) and anti-ERK2 antibody (lower panel), respectively. Arrowheads indicate phosphorylated ERK1/2. B, After mechanical stretch (-21 kPa) for the indicated times, cell lysates were immunoprecipitated with anti-CAKß antibody and then immunoblotted with anti-pTyr antibody (upper panel), anti-FAK antibody (middle panel), and anti-CAKß antibody (lower panel), respectively. An arrowhead denotes FAK (upper and middle panels) and CAKß (lower panel), respectively. C, Cells pretreated with and without cytochalasin D in the indicated concentration for 15 min were stimulated with mechanical stretch (-21 kPa) for 1 min. Cell lysates were immunoprecipitated with anti-CAKß antibody and then immunoblotted with anti-pTyr antibody (upper panel) and anti-CAKßantibody (lower panel), respectively. An arrowhead indicates tyrosine-phosphorylated CAKß.

 
It has been previously demonstrated that CAKß directly cross-phosphorylates FAK, which acts as a target molecule for the SH2 domain-containing proteins (24). To reveal whether mechanical stretch stimulates the association of CAKß with FAK, we examined immunoprecipitation with anti-FAK antibody, followed by immunoblotting with anti-pTyr, anti-FAK, or anti-CAKß antibody. The enhanced phosphorylation of FAK under basal conditions was neither augmented nor associable with CAKß (Fig. 6BGo). Therefore, it is unlikely that FAK plays any role in the CAKß-dependent signaling pathway by mechanical stretch.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study has demonstrated for the first time that mechanical stretch markedly induced activation of CAKß in rat VSMC. Although the exact underlying mechanism(s) of CAKß activation after stretching has not been fully determined, the stimulation of CAKß phosphorylation shared in common by mechanical stretch and Ca2+ ionophore strongly suggests the pivotal role of [Ca2+]i in CAKß activation. The present study has clearly shown that the cell-permeable Ca2+ chelator (BAPTA-AM) completely and the SA channel blocker (Gd) largely blocked the stretch-induced phosphorylation of CAKß in rat VSMC. Furthermore, extracellular Ca2+ chelation by EGTA reduced stretch-induced CAKß phosphorylation, suggesting that Ca2+ influx from extracellular sources via SA ion channels plays an important role in the stretch-induced activation of CAKß. Our data are in good agreement with those from a previous study showing that tyrosine phosphorylation by stretching was mediated by an increase in [Ca2+]i via Gd-sensitive SA ion channels in endothelial cells (25).

The partial inhibition of the stretch-induced CAKß activation by EGTA suggests that extracellular ions distinct from Ca2+ signal(s) may also be involved, because certain SA channels have also been shown to be permeable to Na+ and K+ in VSMC (16). A previous study revealed that mechanical stretch of VSMC induced a transient increase in [Na+]i, which was inhibited by Gd, but not by an L-type Ca2+ channel inhibitor (26). Because [Na+]i is 5–6 orders of magnitude greater than [Ca2+]i, even a slight increase in [Na+]i caused by stretching can be amplified into an intracellular Ca2+ signal sufficient for CAKß activation. In fact, the present study has shown that the effect of a Na+ ionophore on phosphorylation of CAKß was almost comparable to that of a Ca2+ ionophore. Collectively, we propose that an increase in [Na+]i via SA channels is also capable of initiating CAKß activation in response to stretching.

In the present study the SA channel blocker (Gd) did not completely inhibit stretch-induced CAKß phosphorylation. The finding suggests that stretch-induced CAKß phosphorylation may be mediated partially through a mechanism other than SA channel activation.

Accumulating lines of evidence have shown that mechanical stress releases several active factors to activate ERK1/2 in an autocrine/paracrine manner, such as AngII (19) and endothelin-1 (27) from cardiac myocytes, and platelet-derived growth factor (28), fibroblast growth factor (29), and ATP (30) from VSMC. Recently, we demonstrated that AngII stimulated CAKß via an increase in [Ca2+]i in rat VSMC (17). Therefore, it is possible that a diffusable factor(s) secreted from VSMC by stretching stimulates CAKß activation. However, treatment of unstretched control VSMC with the conditioned medium after stretching did not affect phosphorylation of ERK1/2 (our unpublished observation). Furthermore, AT1 receptor antagonist (CV11974) failed to inhibit stretch-induced CAKß activation. Although the possible autocrine/paracrine mechanism by a diffusable factor(s) accumulated after longer incubation or rapidly degraded and/or an otherwise nontransferable factor(s) could not be excluded, the stretch-induced activation of CAKß cannot wholly account for the autocrine/paracrine effect by AngII on tyrosine phosphorylation. Thus, cell and tissue type-specific roles of another diffusable factor(s) in stretch-induced signaling events remain to be determined.

Actin cytoskeleton, which maintains the association of FAK with cell surface integrins or other cytoskeleton components, is essential for FAK phosphorylation at focal adhesion. As the carboxyl-terminal portion of CAKß shows high homology to the focal adhesion-targeting domain of FAK, CAKß may be involved in focal adhesion and cytoskeletal signaling. In fact, it has been reported that histamine induced translocation of CAKß to focal adhesion and activated ERK1/2 via its translocation-dependent mechanism (23). However, the present study revealed that disruption of actin stress fibers with cytochalasin D to dissociate CAKß from focal adhesion failed to inhibit the stretch-induced phosphorylation of CAKß and ERK1/2. Therefore, stretch-induced activation of CAKß and ERK1/2 is unlikely to involve the actin cytoskeleton network.

FAK, one of the important cytosolic tyrosine kinases in mediation of integrin signaling pathways, has been shown to increase its kinase activity and tyrosine phosphorylation by mechanical stimuli in a variety of cells (31). As CAKß directly cross-phosphorylated FAK, which acted as a target for the SH2 domain-containing proteins (24), FAK might modulate CAKß activation by promoting kinase substrate interaction. However, the present study has shown that mechanical stretch failed to enhance the association of FAK with CAKß, and that the enhanced tyrosine phosphorylation under basal conditions was minimally affected by stretching under quiescent and adherent conditions. These data lend strong support to the contention that the stretch-induced activation of CAKß is unrelated to FAK. Consistent with these results, it has been shown that activation of CAKß is more rapid and greater than that of FAK in the cells that expressed both enzymes (10, 11).

CAKß, when autophosphorylated on tyrosine residues, provides binding sites for other SH2 domain-containing proteins, including Src tyrosine kinase family. It is well recognized that the association of CAKß with c-Src leads to translocation of adaptor proteins (Shc/Grb2) to the plasma membrane and subsequent p21Ras-dependent ERK1/2 activation (20, 21). In the present study we confirmed that mechanical stretch stimulated formation of the CAKß/c-Src complex. We recently demonstrated that mechanical stretch induces p21Ras-dependent ERK1/2 activation in rat VSMC (8). Furthermore, in the present study PP2, a potent and selective inhibitor for Src kinase family, significantly inhibited stretch-induced ERK1/2 activation without affecting stretch-induced CAKß phosphorylation. Taken together, ERK1/2 is a possible candidate for a downstream target in the stretch-stimulated CAKß-dependent pathway.

Although the exact role of tyrosine phosphorylation of CAKß in stretch-stimulated signaling remains to be determined, a reversible phosphorylation of tyrosine kinases is known to be a regulatory machinery in cell growth and mitosis (32). In this regard, it has been shown that mechanical strain causes cell proliferation by modulating transcription factors via the ERK1/2 pathway (6). Our previous study has revealed that Ca2+-dependent ERK1/2 activation, mainly via SA ion channels, is required for the stretch-induced hypertrophy of VSMC (8). Based on the observations that mechanical stress to VSMC is enhanced in hypertension, the stretch-stimulated activation of CAKß may play a pivotal role in the process of vascular remodeling by hypertension.

In conclusion, we demonstrated herein that mechanical stretch activates CAKß, which requires ion influx mainly via Gd-sensitive SA channels, thereby leading to ERK1/2 cascade and cell growth in rat VSMC. In this regard, c-Src appears to be a downstream signaling molecule with an ability to bind to CAKß.


   Footnotes
 
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan, and a fund from the Tokyo Hypertension Conference.

Abbreviations: AngII, Angiotensin II; AT1 receptor, angiotensin II type 1 receptor; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester); ([Ca2+]I, intracellular Ca2+ concentration; CAKß, cell adhesion kinase-ß; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; Gd, gadolinium; LPA, lysophosphatidic acid; [Na2+]i, intracellular Na+ concentration; PMA, phorbol 12-myristate 13-acetate; PP-2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; pTyr, phosphotyrosine; pyk2; praline-rich tyrosine kinase 2; SA, stretch activated; Src, tyrosine protein kinase pp60-c-Src; VSMC, vascular smooth muscle cell.

Received September 5, 2002.

Accepted for publication January 31, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Elkind MS, Sacco RL 1998 Stroke risk factors and stroke prevention. Semin Neurol 18:429–440[Medline]
  2. Stamler J, Stamler R, Neaton JD 1993 Blood pressure, systolic and diastolic, and caridiovascular risks: US population data. Arch Intern Med 153:598–615[Abstract]
  3. Hall WD 1999 Risk reduction associated with lowering systolic blood pressure: review of clinical trial data. Am Heart J 138:225–230[CrossRef][Medline]
  4. Carallo C, Irace C, Pujia A, Serena M, De Franceschi S, Crescenzo A, Motti C, Cortese C, Mattioli PL, Gnasso A 1999 Evaluation of common carotid hemodynamic forces: relations with wall thickening. Hypertension 34:217–221[Abstract/Free Full Text]
  5. Jiang Y, Kohara K, Hiwada K 2000 Association between risk factors for atherosclerosis and mechanical forces in carotid artery. Stroke 31:2319–2324[Abstract/Free Full Text]
  6. Lehoux S, Tedgui A 1998 Signal transduction of mechanical stresses in the vascular wall. Hypertension 32:338–345[Abstract/Free Full Text]
  7. Sigurdson W, Ruknudin A, Sachs F 1992 Calcium imaging of mechanically induced fluxes in tissue-cultured chick heart: role of stretch-activated ion channels. Am J Physiol 262:H1110–H1115
  8. Iwasaki H, Eguchi S, Ueno H, Marumo F, Hirata Y 2000 Mechanical stretch stimulates growth of vascular smooth muscle cells via epidermal growth factor receptor. Am J Physiol 278:H521–H529
  9. Avraham H, Park SY, Schinkmann K, Avraham S 2000 RAFTK/Pyk2-mediated cellular signaling. Cell Signal 12:123–133[CrossRef][Medline]
  10. Zheng C, Xing Z, Bian ZC, Guo C, Akbay A, Warner L, Guan J-L 1998 Differential regulation of Pyk2 and focal adhesion kinase (FAK). J Biol Chem 273:2384–2389[Abstract/Free Full Text]
  11. Brinson AE, Harding T, Diliberto PA, He Y, Li X, Hunter D, Herman B, Earp HS, Graves LM 1998 Regulation of a calcium-dependent tyrosine kinase in vascular smooth muscle cells by angiotensin II and platelet-derived growth factor. Dependence on calcium and the actin cytoskeleton. J Biol Chem 273:1711–1718[Abstract/Free Full Text]
  12. Sieg DJ, Ilic D, Jones KC, Damsky CH, Hunter T, Schlaepfer DD 1998 Pyk2 and Src-family protein kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration. EMBO J 17:5933–5947[CrossRef][Medline]
  13. Banes AJ, Gilbert J, Taylor D, Monbureau O 1985 A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro. J Cell Sci 75:35–42[Abstract]
  14. Banes AJ, Link GW, Gilbert JW, Monbureau O 1990 Culturing cells in a mechanically active environment: the Flexer Strain Unit can apply cyclic or static tension or compression to cells in culture. Am Biotechnol Lab 8:12–22[Medline]
  15. Iwasaki H, Shichiri M, Marumo F, Hirata Y 2001 Adrenomedullin stimulates proline-rich tyrosine kinase 2 in vascular smooth muscle cells. Endocrinology 142:564–572[Abstract/Free Full Text]
  16. Meininger GA, Davis MJ 1992 Cellular mechanisms involved in the vascular myogenic response. Am J Physiol 263:H647–H659
  17. Eguchi S, Iwasaki H, Inagami T, Numaguchi K, Yamakawa T, Motley ED, Owada KM, Marumo F, Hirata Y 1999 Involvement of PYK2 in angiotensin II signaling of vascular smooth muscle cells. Hypertension 33:201–206[Abstract/Free Full Text]
  18. Arora PD, Bibby KJ, McCulloch CAG 1994 Slow oscillations of free intracellular calcium ion concentration in human fibroblasts responding to mechanical stretch. J Cell Physiol 161:187–200[CrossRef][Medline]
  19. Sadoshima J, Xu Y, Slayter HS, Izumo S 1993 Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell 75:977–984[CrossRef][Medline]
  20. Dikic I, Tokiwa G, Lev S, Courtneidge SA, Schlessinger J 1996 A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation. Nature 383:547–550[CrossRef][Medline]
  21. Blaukat A, Ivankovic-Dikic I, Grönroos E, Dolfi F, Tokiwa G, Vuori K, Dikic I 1999 Adaptor proteins Grb2 and Crk couple Pyk2 with activation of specific mitogen-activated protein kinase cascades. J Biol Chem 274:14893–14901[Abstract/Free Full Text]
  22. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA 1996 Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor. J Biol Chem 271:695–701[Abstract/Free Full Text]
  23. Litvak V, Tian D, Shaul YD, Lev S 2000 Targeting of PYK2 to focal adhesions as a cellular mechanism for convergence between integrins and G protein-coupled receptor signaling cascades. J Biol Chem 275:32736–32746[Abstract/Free Full Text]
  24. Li X, Dy RC, Cance WG, Graves LM, Earp HS 1999 Interactions between two cytoskeleton-associated tyrosine kinases: calcium-dependent tyrosine kinase and focal adhesion tyrosine kinase. J Biol Chem 274:8917–8924[Abstract/Free Full Text]
  25. Naruse K, Yamada T, Sai XR, Hamaguchi M, Sokabe M 1998 Pp125FAK is required for stretch dependent morphological response of endothelial cells. Oncogene 17:455–463[CrossRef][Medline]
  26. Liu X, Hymel LJ, Songu-Mize E 1998 Role of Na+ and Ca2+ in stretch-induced Na+-K+-ATPase {alpha}-subunit regulation in aortic smooth muscle cells. Am J Physiol 274:H83–H89
  27. Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I, Hiroi Y, Mizuno T, Maemura K, Kurihara H, Aikawa R, Takano H, Yazaki Y 1996 Endothelin-1 is involved in mechanical stress-induced cardiomyocytes hypertrophy. J Biol Chem 271:3221–3228[Abstract/Free Full Text]
  28. Wilson E, Mai Q, Sudhir K, Weiss RH, Ives HE 1993 Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF. J Cell Biol 123:741–747[Abstract/Free Full Text]
  29. Cheng GC, Libby P, Grodzinsky AJ, Lee RT 1996 Induction of DNA synthesis by a single transient mechanical stimulus of human vascular smooth muscle cells. Role of fibroblast growth factor-2. Circulation 93:99–105[Abstract/Free Full Text]
  30. Hamada K, Takuwa N, Yokoyama K, Takuwa Y 1998 Stretch activates Jun N-terminal kinase/stress-activated protein kinase in vascular smooth muscle cells through mechanisms involving autocrine ATP stimulation of purinoceptors. J Biol Chem 273:6334–6340[Abstract/Free Full Text]
  31. Cary LA, Guan J-L 1999 Focal adhesion kinase in integrin-mediated signaling. Front Biosci 4:d102–d113
  32. Luttrell LM, Daaka Y, Lefkowitz RJ 1999 Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol 11:177–183[CrossRef][Medline]



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